Industrial applications of vaccines: Vaccines are considered to be among the most cost-effective and health-preserving medical inventions ever developed. The rationale for vaccination is that pre-exposure of the host to a vaccine against a given infectious agent can ameliorate or prevent disease should the vaccinated individual become exposed to that agent at a later time. The gap in time between vaccination and possible exposure requires “memory” on the part of the immune system. This memory is embodied in the persistence of immune cells for years or even decades after vaccination. Creating vaccines that induce strong and lasting protection is a difficult task, given our incomplete knowledge of the immune system. Nevertheless, continuing advances in our understanding make possible new approaches to vaccine design.
Vaccines against infectious agents: For microbial agents, many vaccines in use are comprised of live attenuated or non-virulent strains of the disease-causing microorganisms. Other vaccines are comprised of killed or otherwise inactivated microorganisms. Yet other vaccines utilize purified components of pathogen lysates, such as surface carbohydrates or recombinant pathogen-derived proteins. Vaccines that utilize live attenuated or inactivated pathogens typically yield a vigorous immune response, but their use has limitations. For example, live vaccine strains can sometimes mutate back into disease-causing variants, especially when administered to immunocompromised recipients. Moreover, many pathogens, particularly viruses, undergo continuous rapid mutations in their genome, which allow them to escape immune responses to antigenically distinct vaccine strains.
Vaccines for the prevention or treatment of cancer: As the understanding of immunity has developed, it became clear that the immune system also controls or attempts to control the development of malignancies (Dunn et al., 2002; 3(11):991-8). As a result, immunotherapy is now being used to eradicate or control certain human cancers. Some of the technology and concepts of vaccines against infectious agents also apply to using the immune system to fight cancers, both solid tumors and blood cancers such as leukemia. Patients at risk for cancer, such as those infected by cancer-associated viruses like human papilloma virus (HPV), can be protected from developing the particular cancer in question as exemplified by Gardasil® vaccination against human papilloma virus (HPV), which causes cervical cancer. Patients who already have cancer, such as prostate cancer, can also be helped by vaccination, as exemplified by the Provenge® vaccine which is an immunotherapy for prostate cancer.
CD8+ T cells can recognize conserved antigens in many infectious agents and prevent disease: While these have been successful vaccines, there have been major problems constructing vaccines against antigens from rapidly mutating infectious agents such as influenza, HIV, and Plasmodium falciparum (a cause of malaria). In these cases and others, the infectious agent has surface protein(s) that can rapidly mutate to evade otherwise protective antibodies. Nevertheless, these agents also have relatively conserved and unchanging internal components as exemplified by nucleoprotein (NP) of influenza, Gag and Pol for HIV, and circumsporozoite surface protein (CSP) for Plasmodium falciparum. In these cases, antibodies (which can only bind to the surface of pathogens) are unable to bind to these more conserved and internal antigens. Instead, there is a well-established role for CD8+ T cells in controlling or clearing such infectious agents—provided that a strong enough CD8+ T cell response can be generated. To cite just three examples: (1) Protection from disease caused by influenza can be achieved by high levels of CD8+ T cells against the conserved nucleoprotein (NP) viral protein (Webster et al., Eur J Immunol. 1980; 10(5):396-401; Slutter et al., J Immunol. 2013; 190(8):3854-8. PMCID: 3622175.). (2) Strong CD8+ T cell responses against the Gag and Pol proteins of simian immunodeficiency virus (SIV, a non-human primate model for HIV infection) can protect macaques from developing AIDS after challenge with SIV (Hansen et al., Nature. 2011; 473(7348):523-7. PMCID: 3102768). (3) CD8+ T cells against Plasmodium falciparum antigens can protect humans from malaria (Epstein et al., Science. 2011; 334(6055):475-80). Thus, there is an urgent and largely unmet need to develop better ways of eliciting strong CD8+ T cells to protect against infection.
CD8+ T cells can recognize cancer antigens and cure malignancy: Similar to the situation with infectious agents, CD8+ T cells can also be generated against tumor cell antigens. As exemplified by Tumor-Infiltrating Lymphocytes (TILs), the passive administration of anti-tumor CD8+ T cells can be sufficient to cure patients of advanced cancers in a small percentage of cases (Restifo et al., Nature Reviews Immunology. 2012; 12(4):269-81). These CD8+ T cells recognize peptides termed “tumor antigens” where the tumor contains antigens either not found in normal tissue or present at much lower levels. As noted above, some tumor antigens are derived from tumorigenic viruses such as the E6 and E7 antigens in HPV-related cervical cancer. Other tumor antigens are derived from mutations in germline proteins such as the V600E mutation in the BRAF protein. Yet other tumor antigens are normal proteins such as HER-2/neu which is overexpressed in breast cancer, where the breast is a non-essential “disposable” tissue that can be sacrificed by an immune attack on breast-derived tissues. Here again, there is an urgent and largely unmet need to develop better ways of eliciting strong CD8+ T cell responses to protect against cancer or treat patients with already established malignant disease.
Numerous licensed vaccines are live, attenuated viruses (LAV): As noted above, there is a major problem in the art which is that it has been difficult to develop industrial applicable vaccines that are able to generate antigen-specific CD8+ T cells. For viral infections, one of the best ways is to generate anti-viral CD8+ T cells is to vaccinate with a live, attenuated virus (LAV) vaccine. Familiar examples of LAV vaccines are the Measles/Mumps/Rubella (MMR) vaccine, Sabin poliovirus vaccine, FluMist® influenza vaccine, Yellow Fever Virus 17D vaccine, and Vaccinia smallpox vaccine. But it has been difficult to produce LAV vaccines against viral infections for a variety of reasons that include inefficient manufacturing process, a need for repeated vaccination with follow-up “booster” vaccination many years later, and the generally poor quality and low level of the CD8+ T cell response to many vaccine candidates.
CD8+ T cells can cure cancer in humans but are difficult to generate: For cancers not associated with viruses, there is no possibility of developing an LAV type vaccine. Instead, tumor antigens must be identified or otherwise isolated or predicted and used for vaccination. To be curative for cancer, a substantial CD8+ T cell response is needed. This has been shown for regimens that isolate and expand tumor-infiltrating lymphocytes (TIL) which are CD8+ T cells grown ex vivo and then administered back to the patients. In these studies, a relatively high number of TIL CD8+ T cells is required to successfully eradicate and cure metastatic melanoma (Restifo et al., Nature Reviews Immunology. 2012; 12(4):269-81). Many seemingly auspicious cancer vaccines and immunotherapies turn out to be too weak to cure cancer when tested in vivo. For example, simply vaccinating with a tumor antigen peptide emulsified in Montanide lipid as an immunostimulant fails to cure cancer because the resulting CD8+ T cells do not enter the circulation and go to the tumors (Hailemichael et al., Nat Med. 2013; 19(4):465-72. PMCID: 3618499).
CD8+ T cells are stimulated by antigen peptides presented on MHC Class I (MHC-I): In order to understand the process for generating CD8+ T cells, it is helpful to review how they arise during a normal immune response. CD8+ T cells are named because they have the CD8 protein on their surface. CD8 works as a “co-receptor” along with the T cell receptor (TCR) to recognize peptide antigens (typically 7-11 amino acids in length) that are processed inside of cells by the cleavage of the intact proteins and then displayed on the surface of infected cells by major histocompatibility complex (MHC) Class I (MHC-I) molecules. These MHC-I molecules hold the peptide antigen in a “groove” and the CD8+ T cell then recognizes the peptide-MHC-I (pMHC-I) complex and becomes activated. CD8+ T cells that kill the infected cell are termed “cytotoxic” but they can also interfere with infectious agents by producing cytokines such as interferon-gamma (IFN-g).
Considering the foregoing, it is highly desirable to find an industrially applicable means for producing vaccines that are highly effective for eliciting strong CD8+ T cells, CD4+ T cells, and antibody responses against infectious agents and tumor antigens.
Need for antigen-presenting cells (APC) to generate antigen-specific CD8+ T cells: With this as an introduction, it can be appreciated that a key event in the generation of CD8+ T cells is to develop a cell type called an “antigen-presenting cell” (APC) that can present pMHC-I to uneducated or naïve CD8+ T cell precursors to induce them to divide, expand in numbers, and persist for prolonged periods as highly active “memory” CD8+ T cells. To be effective at generating CD8+ T cells, an APC must both express peptide antigen on MHC-I (pMHC-I) that is recognized by the TCR (called “Signal 1”) and also co-stimulate the responding cells through additional receptor (called “Signal 2”) and even other receptors (called “Signal 3”). TCR stimulation by pMHC-I provides Signal 1 and generally stimulation of the CD28 receptor on CD8+ T cells provides Signal 2. Signal 3 can be provided in a non-redundant fashion either by soluble proteins such as interferon-alpha (Type I interferon) and/or interleukin-12 (IL-12) and/or cell surface molecules such as CD27 ligand (CD27L, also called CD70 or TNFSF7), 4-1BBL (also called CD137L or TNFSF9), and/or OX40L (also called CD134L or TNFSF4) (Sanchez and Kedl, Vaccine. 2012; 30(6):1154-61. PMCID: 3269501). What is needed is a vaccine approach that can activate an APC to provide all of these signals. This requires a good dendritic cell stimulus, also called an “immune adjuvant” or “adjuvant.”
APC cross-presentation of extracellular antigens: The first requirement for an APC is to express peptide antigen on MHC-I (pMHC-I). The prototypic APC is the dendritic cell which takes up protein antigens from its environment, degrades these proteins into peptides, loads the resulting peptides onto MHC-I, and then presents the pMHC-I on their surface to provide the TCR stimulus that is Signal 1. This process is very different from cells infected by a microbial pathogen or tumor cells. In those cases, the protein antigen is produced within the cell itself—not taken up from the extracellular space—and then protein degradation products (which are peptides) are loaded onto MHC-I and exported to the cell surface as pMHC-I to provide Signal 1. What makes dendritic cells and other APCs special is that they can form pMHC-I from proteins in their environment, a phenomenon termed “cross-presentation.” For dendritic cells to do this, they must take up the protein antigen from their environment using one of a few very specialized receptors, including DEC205, CD11c, BDCA1, BDCA3, and/or CD40. After taking up protein antigen from the extracellular space, these receptors direct the delivery of the protein antigen into membrane-limited intracellular compartments (“endosomes”) where the protein can be digested into peptides and then transferred into compartments where MHC-I is being assembled. Of special important to the instant invention is that the best receptor on dendritic cells for processing protein antigen into pMHC-I (i.e., crosspresentation) is the CD40 receptor (Chatterjee et al., Blood. 2012; 120(10):2011-20; Cohn et al., J Exp Med. 2013; 210(5):1049-63. PMCID: 3646496). Therefore, it is highly desirable for a vaccine to include a protein antigen that is targeted toward the CD40 receptor on dendritic cells.
Activation of the APC stimulates crosspresentation: A second requirement for an APC to crosspresent an exogenous protein antigen is for the APC to be “activated.” For dendritic cells, such activation is ideally provided by an effective stimulus through the CD40 receptor, which promotes crosspresentation and the formation of the pMHC-I Signal 1 (Delamarre et al., J Exp Med. 2003; 198(1):111-22). Similarly, B cells, which are another type of APC, can be activated by a CD40 receptor stimulus to crosspresent soluble protein antigens (Ahmadi et al., Immunology. 2008; 124(1):129-40).
Crosspresentation of antigen by dendritic cells in the absence of CD40 stimulation leads to CD8+ T cell tolerance: DEC-205 is a receptor on dendritic cells and B cells recognized on mouse cells by the NLDC-145 monoclonal antibody (Inaba et al., Cellular immunology. 1995; 163(1):148-56). Bonifaz et al. showed that the binding portion of an anti-DEC205 antibody can be genetically fused to a model antigen, chicken ovalbumin (OVA). The injection of anti-DEC205/OVA fusion protein directs the OVA antigen to dendritic cells and leads to crosspresentation of OVA peptide antigen on MHC-I. However, while this treatment induces anti-OVA CD8+ T cells to divide and proliferate, these cells soon die off and are deleted. This results in specific tolerance for OVA that cannot be overcome by subsequent vaccination with OVA plus Complete Freund's Adjuvant (CFA), which is usually considered to be a gold standard for vaccination (although CFA is far too inflammatory to be used in humans). However, if anti-DEC205/OVA fusion protein is combined with a stimulus for the CD40 receptor, then very strong anti-OVA CD8+ T cell responses result (Bonifaz et al., J Exp Med. 2002; 196(12):1627-38. PMCID: 2196060). This indicates that simply targeting antigens to dendritic cells alone (e.g., using a fusion protein of anti-DEC205 and antigen) does not succeed in eliciting high levels of efficacious and persisting antigen-specific CD8+ T cells. In fact, it shows that allowing antigen to be taken up by unactivated dendritic cells should be avoided because it will work against the goal of creating strong antigen-specific CD8+ T cell responses.
Generating CD8+ T cell responses is best when antigen is delivered to dendritic cells in conjunction with an adjuvant: Although they did not use a CD40 stimulus, Kamath et al. (J Immunol. 2012; 188(10):4828-37) developed a vaccine system for delivering an antigen either directly attached to an antigen or co-delivered with a separate, unattached antigen. When antigen was delivered to DCs in the absence of adjuvant, antigen-specific T cells were induced to proliferate but did not subsequently differentiate into effector cells. Instead, effective immunity was only induced when the test vaccine provided antigen and adjuvant to the same individual DCs within a short window of time. These parameters are fulfilled when the antigen and adjuvant are linked in time and space as parts of the very same molecule, as provided by the instant invention.
To fulfill the need for a vaccine that induces a strong CD8+ T cell responses, the instant invention provides for a composition that contains, for example, CD40 ligand (CD40L, TNFSF5, which is an agonist of the CD40 receptor) physically linked to a multimerization domain that organizes it into a highly active many-trimer structure in addition to being physically linked to an antigen. In this way, antigen can be targeted to dendritic cells via binding to the CD40 receptor on their surface and activates the dendritic cell simultaneously. This arrangement can thus avoid delivery of antigen to dendritic cells that do not become activated and which instead would induce antigen-specific CD8+ T cell tolerance. As a result, the compositions of the instant invention provide for a unusually high level of activity in inducing strong CD8+ T cell responses, where the TCRs of elicited CD8+ T cells show an exceptionally high level of avidity for pMHC-I and where a vaccine of the invention confers surprisingly profound protection from challenge by an infectious agent (Vaccinia encoding HIV-1 Gag as a model antigen). Variations on these compositions are expected to elicit very strong CD4+ T cells and B cell antibody responses in a similar fashion.